Molecular characterization of an endophytic strain of Bacillus subtilis with plant growth promoting properties from a wild relative of Papaya.

Aim: Bacillus subtilis is usually found in soil, and their biocontrol and plant growth promoting capabilities are being explored more recently than ever. However, knowledge about metabolite production and genome composition of endophytic Bacillus subtilis from seeds is limited. In the present study, Bacillus subtilis EVCu15 strain isolated from the seeds of Vasconcellea cundinamarcensis (mountain papaya) was subjected to whole genome sequencing, and detailed molecular and functional characterization.

Methods And Results: Whole genome sequencing and sequence analysis of the endophytic bacterium from mountain papaya seed revealed that the bacterium was Bacillus subtilis, strain EVCu15. The genomic sequence had more than 98% nucleotide similarity with two published whole genome sequences of Bacillus subtilis strains. Some of the important secondary metabolite gene clusters involved in production of bioactive compounds such as surfactin, fengycin, plipastatin, bacillibactin, bacillaene, subtilomycin, subtilosin A and bacilysin were identified from the whole genome sequence analysis. Genes encoding several plant growth-promoting metabolites, mostly involved in the nutrient metabolism, were identified in the bacterial genome. These included factors coding for nitrogen, phosphorus, iron, sulfur, potassium, and trehalose metabolism. Genes involved in auxin, riboflavin, acetoin biosynthesis, ACC deaminase activity and xylan degradation were also identified. Proteomic analysis confirmed the biosynthesis and release of several bioactive secondary metabolites in the endophytic B. subtilis strain EVCu15. LCMS-based profiling for hormones and vitamins identified extracellular secretion of several important plant growth-promoting compounds such as IAA, salicylic acid, zeatin, vitamin D1, D2, E, K1 and pyridoxine. The in vitro and in vivo studies with the endophytic B. subtilis against various plant pathogenic fungi showed moderate to high levels of resistance. The B. subtilis EVCu15 compared to B. amyloliquefaciens showed better control over the root knot nematode Meloidogyne incognita, in terms of egg hatching inhibition and the mortality of J2 juveniles.

Conclusion: Overall, this study underscores the biocontrol and plant growth-promoting potential of Bacillus subtilis EVCu15, an endophyte isolated from mountain papaya seeds. Genomic analysis revealed a significant proportion of genes linked to biocontrol and plant growth promotion, corroborating its efficacy against Meloidogyne incognita and various plant pathogens in vitro and in greenhouse studies. Furthermore, the bacterium's ability to produce diverse bioactive compounds, including proteins, hormones, and vitamins, was confirmed, highlighting its complex interactions within the plant system.